William K Rashbaum

Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain

Both late-gestation and adult human forebrain contain large numbers of oligodendrocyte progenitor cells (OPCs). These cells may be identified by their A2B5+PSA-NCAM− phenotype (positive for the early oligodendrocyte marker A2B5 and negative for the polysialylated neural cell adhesion molecule). We used dual-color fluorescence-activated cell sorting (FACS) to extract OPCs from 21- to 23-week-old fetal human forebrain, and A2B5 selection to extract these cells from adult white matter. When xenografted to the forebrains of newborn shiverer mice, fetal OPCs dispersed throughout the white matter and developed into oligodendrocytes and astrocytes. By 12 weeks, the host brains showed extensive myelin production, compaction and axonal myelination. Isolates of OPCs derived from adult human white matter also myelinated shiverer mouse brain, but much more rapidly than their fetal counterparts, achieving widespread and dense myelin basic protein (MBP) expression by 4 weeks after grafting. Adult OPCs generated oligodendrocytes more efficiently than fetal OPCs, and ensheathed more host axons per donor cell than fetal cells. Both fetal and adult OPC phenotypes mediated the extensive and robust myelination of congenitally dysmyelinated host brain, although their differences suggested their use for different disease targets.

High-yield selection and extraction of two promoter-defined phenotypes of neural stem cells from the fetal human brain

Neural stem and precursor cells reside in the ventricular lining of the fetal forebrain, and may provide a cellular substrate for brain repair. To selectively identify and extract these cells, we infected dissociated fetal human brain cells with adenoviruses bearing the gene for green fluorescence protein (GFP), placed under the control of enhancer/promoters for two genes (nestin and musashi1) that are expressed in uncommitted neuroepithelial cells. The cells were then sorted by fluorescence-activated cell sorting (FACS) on the basis of E/nestin- or P/musashi1-driven GFP expression. Both P/musashi1:hGFP- and E/nestin:EGFP-sorted cells were multipotent: limiting dilution with clonal expansion as neurospheres, in tandem with retroviral lineage analysis and xenograft to E17 and P0-2 rat forebrain, revealed that each phenotype was able to both self-renew and co-generate neurons and glia. Thus, fluorescent genes placed under the control of early neural promoters allow neural stem cells to be specifically targeted, isolated, and substantially enriched from the fetal human brain.

Telomerase immortalization of neuronally restricted progenitor cells derived from the human fetal spinal cord

Lineage-restricted progenitors of the central nervous system (CNS) are not readily expandable because their mitotic competence is limited. Here we used retroviral overexpression of human telomerase reverse transcriptase (hTERT) to immortalize progenitors from human fetal spinal cord. The hTERT-immortalized cells divided in basic fibroblast growth factor (bFGF) expressed high telomerase activity, and gave rise to phenotypically restricted subpopulations of either glia or neurons. The latter included a prototypic line, hSC11V-TERT, that gave rise only to neurons. These included both chx10+ interneurons and Islet1+/Hb9+/ChAT+ motor neurons; the latter were recognized by green fluorescent protein (GFP) driven by the Hb9 enhancer. The neurons were postmitotic and achieved electrophysiologic competence. Upon xenograft to both fetal rat brain and injured adult spinal cord, they matured as neurons and survived for 6 months, with no evident tumorigenesis. The cells have survived >168 doublings in vitro, with karyotypic normalcy and without replicative senescence. hTERT overexpression thus permits the generation of progenitor lines able to give rise to phenotypically restricted neurons.

Cytokine Preconditioning Promotes Codifferentiation of Human Fetal Liver CD133+ Stem Cells Into Angiomyogenic Tissue

Background—CD133 (AC133) is a surface antigen that defines a broad population of stem cells, including myogenic and endothelial progenitors. CD133+ cells are rare in adult tissues, and the factors that support their differentiation into mature angiomyogenic cells are not known. These hurdles have hampered the use of CD133+ cells for therapeutic purposes. Because human fetal liver is a rich source of CD133+ cells, we sought to identify the growth factors that promote codifferentiation of these cells into angiogenic and myogenic cells. Methods and Results—Human fetal liver CD133+ and CD133− cell subpopulations were cultured with 5′-azacytidine or vascular endothelial growth factor (VEGF165) and/or brain-derived nerve growth factor (BDNF). CD133+ but not CD133− cells from human fetal liver codifferentiated into spindle-shaped cells, as well as flat adherent multinucleated cells capable of spontaneous contractions in culture. The resulting spindle-shaped cells were confirmed to be endothelial cells by immunohistochemistry analysis for von Willebrand factor and by acetylated LDL uptake. Multinucleated cells were characterized as striated muscles by electron microscopy and immunohistochemistry analysis for myosin heavy chain. Presence of VEGF165 and BDNF significantly enhanced angiomyogenesis in vitro. Inoculation of cells derived from CD133+ cells, but not CD133− cells, into the ear pinna of NOD/SCID mice resulted in the formation of cardiomyocytes, as identified by immunostaining with cardiac troponin-T antibody. These cells generated electrical action potentials, detectable by ECG tracing. Conclusions—CD133 defines a population of human fetal liver cells capable of differentiating into both angiogenic and myogenic cells. Preconditioning of these CD133+ cells with VEGF165 and BDNF enhances the angiomyogenesis. CD133+ fetal liver cells ultimately may be used for therapeutic angiomyogenesis.

Fetal Stromal-Dependent Paracrine and Intracrine Vascular Endothelial Growth Factor-A/Vascular Endothelial Growth Factor Receptor-1 Signaling Promotes Proliferation and Motility of Human Primary Myeloma Cells

Induction of neoangiogenesis plays an important role in the pathogenesis of multiple myeloma. However, the mechanism by which expression of vascular endothelial growth factor (VEGF)-A and its receptors modulate the interaction of multiple myeloma cells with stromal cells is not known. Here, we describe a novel in vitro coculture system using fetal bone stromal cells as a feeder layer, which facilitates the survival and growth of human primary multiple myeloma cells. We show that stromal-dependent paracrine VEGF-A signaling promotes proliferation of human primary multiple myeloma cells. Primary multiple myeloma cells only expressed functional VEGF receptor (VEGFR)-1, but not VEGFR-2 or VEGFR-3. VEGFR-1 expression was detected in the cytoplasm and the nuclei of proliferating multiple myeloma cells. Inhibition of VEGFR-1 abrogated multiple myeloma cell proliferation and motility, suggesting that the functional interaction of VEGF-A with its cognate receptor is essential for the growth of primary multiple myeloma cells. Collectively, our results suggest that stromal-dependent paracrine and intracrine VEGF-A/VEGFR-1 signaling contributes to human primary multiple myeloma cell growth and therefore, VEGFR-1 blockade is a potential therapeutic strategy for the treatment of multiple myeloma.

Studies of Transplantation of Human Fetal Tissue in Man

Fetal pancreatic islet tissue has several advantages as a potential transplant tissue for the treatment of patients with Type I diabetes (1–4). We embarked on a series of studies involving procurement, processing, and implantation of fetal pancreatic tissue into Type I insulin-dependent diabetic patients. This chapter reviews these studies.

Studies of human fetal pancreatic allografts in diabetic recipients without immunosuppression

Four Type I insulin-dependent diabetic men received minced tissue from 6-12 pooled fetal pancreata cultured for 48 hours. Immunosuppressive therapy was not given. The tissue was transplanted under local anesthesia into the brachioradialis muscle of the nondominant arm or below the subcutaneous adipose tissue of the left lower quadrant of the abdominal wall. An increase in C-peptide secretion was documented following each procedure. Detectable C-peptide secretory capacity has persisted for 1 year in three cases. The total insulin requirement showed a drop of 71-100% at the time of maximum C-peptide secretion. No increase in anticytoplasmic islet cell antibody titer was detected during the year of observation following transplantation. These studies document that transplantation of functioning fetal pancreatic insulin-secreting tissue can be performed with minimal operative or immunologic risk to the recipient. Significant insulin secretory capacity persists for 1 year following implantation. Further studies are warranted in order to optimize insulin secretion following fetal pancreatic islet transplantation.